Although de novo synthesis of cholesterol occurs in virtually all cells, this capacity is greatest in liver, intestine, adrenal cortex and reproductive tissues, including ovaries, testes and placenta. From an inspection of its structure, it is apparent that cholesterol biosynthesis requires a source of carbon atoms and considerable reducing power to generate the numerous carbon-hydrogen and carbon-carbon bonds. All of the carbon atoms of cholesterol are derived from acetate. Reducing power in the form of NADPH is provided mainly by enzymes of the hexose monophosphate shunt, specifically, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The mevalonate pathway, i.e., the pathway of cholesterol synthesis, occurs in the cytoplasm and is driven in large part by the hydrolysis of the high-energy thioester bonds of acetyl CoA and the high-energy phosphoanhydride bonds of ATP. For a detailed discussion of the mevalonate pathway, see, e.g., Stryer, L., BIOCHEMISTRY, Third Edition (W. H. Freeman And Company/New York (1988)).
The first committed step in the mevalonate pathway is the synthesis of mevalonic acid, which is derived from acetyl CoA. Acetyl CoA can be obtained from several sources: (a) the xcex2 oxidation of long-chain fatty acids; (b) the oxidation of ketogenic amino acids such as leucine and isoleucine; and (c) the pyruvate dehydrogenase reaction. In addition, free acetate can be activated to its thioester derivative at the expense of ATP by the enzyme acetokinase, which is also referred to as acetate thiokinase.
The first two steps in the mevalonate pathway are shared by the pathway that also produces ketone bodies. Two molecules of acetyl CoA condense to form acetoacetyl CoA in a reaction catalyzed by acetoacetyl Coa thiolase (acetyl Coa: acetyl CoA acetyltransferase). The next step introduces a third molecule of acetyl CoA in the cholesterol pathway and forms the branched-chain compound 3-hydroxy-3-methylglutaryl CoA (HMG CoA). This condensation reaction is catalyzed by HMG CoA synthase (3-hydroxy-3-methylglutaryl CoA: acetoacetyl CoA lyase). Liver parenchymal cells contain two isoenzyme forms of HMG CoA synthase; one is found in the cytosol and is involved in cholesterol synthesis, while the other has a mitochondrial location and functions in the pathway that forms ketone bodies. In the HMG CoA synthase reaction, an aldol condensation occurs between the methyl carbon of acetyl CoA and the xcex2-carbonyl group of acetoacetyl CoA with the simultaneous hydrolysis of the thioester bond of acetyl CoA.
The step that produces the unique compound mevalonic acid from HMG CoA is catalyzed by the important microsomal enzyme HMG CoA reductase (mevalonate: NADP+oxidoreductase) that has an absolute requirement for NADPH as the reductant. This reduction reaction is irreversible and produces (R)-(+) mevalonate, which contains six carbon atoms. HMG CoA reductase catalyzes the rate-limiting reaction in the pathway of cholesterol biosynthesis. HMG CoA reductase is an intrinsic membrane protein of the endoplasmic reticulum whose carboxyl terminus extends into the cytoplasm and carries the enzyme""s active site.
Mevalonate is converted into 3-isopentenyl pyrophosphate by three consecutive reactions involving ATP. In the last step, the release of CO2 from 5-pyrophosphomevalonate occurs in concert with the hydrolysis of ATP to ADP and Pi. Thereafter, squalene is synthesized from 3-isopentenyl pyrophosphate by the reaction sequence:
C5xe2x86x92C10xe2x86x92C15xe2x86x92C30
This stage in the mevalonate pathway starts with the isomerization of isopentenyl pyrophosphate to dimethylallyl pyrophosphate. These isomeric C5 units condense to form a C10 compound: an allelic carbonium formed from dimethylallyl pyrophosphate is attacked by isopentenyl pyrophosphate to form geranyl pyrophosphate. The same kind of reaction occurs again: geranyl pyrophosphate is converted into an allelic carbonium ion, which is attacked by isopentenyl pyrophosphate. The resulting C15 compound is called farnesyl pyrophosphate. The last step in the synthesis of squalene is a reductive condensation of two molecules of farnesyl pyrophospate.
The final stage of the mevalonate pathway starts with the cyclization of squalene. This stage, in contrast to the preceding ones, requires molecular oxygen. Squalene epoxide, the reactive intermediate, is formed in a reaction that uses O2 and NADPH. Squalene epoxide is then cyclized to lanosterol by a cyclase. There is a concerted movement of electrons through four double bonds and a migration of two methyl groups in this remarkable closure. Finally, lanosterol is converted into cholesterol by the removal of three methyl groups, the reduction of one double bond by NADPH, and the migration of the other double bond.
From the foregoing, it is apparent that the mevalonate pathway is responsible for the synthesis of a wide variety of essential and clinically relevant molecules. Products from the mevalonate pathway include, for example, cholesterol and the prenyl groups which are required for the function of numerous small GTP binding proteins including the RAS oncoprotein. The connection of the mevalonate pathway, by virtue of its products, to many clinically important processes, such as atherosclerosis and RAS-based cancers, has led to a significant effort to understand the cellular regulation of the mevalonate pathway. It has been found that the mevalonate pathway is regulated by feedback control of several pathway enzymes, the principal one of which is 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol synthesis. Feedback regulation of HMG-CoA reductase occurs through modulation of the steady-state amount of protein by the coordinated adjustment of synthesis and degradation rates. Thus, when the mevalonate pathway products are abundant, the synthetic rate of HMG-CoA reductase is low, the degradation rate of HMG-CoA reductase is rapid, and through both of these actions, the steady state of the enzyme is kept low. Conversely, when production of the mevalonate pathway products is slowed, the synthetic rate of the HMG-CoA reductase is increased, the degradation rate of HMG-CoA reductase is slowed and, consequently, the steady-state level of HMG-CoA reductase is elevated. To date, the molecular signals which control the synthesis and degradation of HMG-CoA reductase are unknown. However, it is known that the control of HMG-CoA reductase degradation is genetically distinct from the control of HMG-CoA reductase synthesis, implying that distinct molecular mechanisms are responsible for these independent features of HMG-CoA reductase regulation.
The feedback regulation of the mevalonate pathway plays a central role in the clinical management of hypercholesterolemia. The current drugs of choice for lowering serum cholesterol are the structurally related, competitive inhibitors of HMG-CoA reductase which include, for example, the widely used Mevacor(copyright), the tradename for lovastatin. These agents were developed specifically for the purpose of inhibiting HMG-CoA reductase. HMG-CoA reductase was thought to be the best target for clinical control of the pathway because this enzyme is most strongly regulated in the physiological control of the cholesterol pathway. However, when a patient is put on an inhibitor of HMG-CoA reductase, the coordinated feedback regulation of HMG-CoA reductase synthesis and degradation brings about a compensatory increase of HMG-CoA reductase levels which abrogates most of the effect of the drug on body cholesterol synthesis. The beneficial effects of HMG-CoA reductase inhibitors, such as Mevacor(copyright), are thought to occur by increased clearance of plasma LDL brought about by the parallel up-regulation of the LDL receptor (LDL-R). Thus, the feedback signals from the mevalonate pathway severely limit the efficacy of such treatments on cholesterol synthesis, but, at the same time, are necessary for the beneficial effects of cholesterol clearing caused by drugs such as Mevacor(copyright).
From the foregoing, it is apparent that the potentially simple solution of interfering with the feedback signals from the mevalonate pathway in order to block the up-regulation of HMG-CoA reductase would also remove the beneficial increase of LDL receptors brought about by the same signals. Moreover, although the possibility exists that the detrimental up-regulation of HMG-CoA reductase synthesis could be specifically blocked without affecting the beneficial up-regulation of the LDL-R, the mechanistic similarity of the two processes may make this separation an unrealistic goal. As such, there remains a need in the art for appropriate molecular targets which can be used to manipulate the levels of HMG-CoA reductase in a manner which is independent of the beneficial LDL receptor control axis.
Another problem in the art is the function of many nucleic acid and polypeptide sequences on deposit in various sequence repositories. For example, one good system for studying the regulation of HMG-CoA is yeast, which encode an HMG-CoA reductase homologue. However, despite the fact that the entire yeast genome has been sequenced and the sequences deposited in GenBank(trademark) and other sequence repositories, the relationship of many yeast genes to HMG-CoA reductase regulation is unknown. Elucidation of the complete yeast genome in the absence of functional information for a particular yeast gene is insufficient for identification of any particular gene product. Although many open reading frames (ORFs) have been identified, it is often not known whether these ORFs encode functional mRNAs, or what the function of the putative genes would be. Similarly, many eukaryotic sequences (e.g., human sequences) have been deposited in GenBank(trademark) and other sequence repositories without any functional information for the sequences being available. In the absence of knowledge regarding the function of a given sequence, there is no reason to select the sequences for cloning into vectors, e.g., for expression of the nucleic acids and encoded proteins. In the absence of functional information, any relationship between deposited sequences and HMG-CoA reductase is unknown.
The present invention provides isolated nucleic acid sequences which encode a family of HMG-CoA Reductase Degradation (HRD) polypeptides. More particularly, the present invention provides isolated HRD1, HRD2 and HRD3 nucleic acids, vectors which replicate and/or express the nucleic acids, and Hrd polypeptides encoded by the nucleic acids, i.e., Hrd1p, Hrd2p and Hrd3p, respectively. It is now discovered that the Hrd polypeptides of the present invention regulate the degradation of HMG-CoA reductase, the enzyme which catalyzes the rate-limiting reaction in the pathway of cholesterol biosynthesis. As such, the Hrd polypeptides of the present invention can be used to regulate the degradation of HMG-CoA reductase. Moreover, the Hrd polypeptides of the present invention can be used in various assays to identify other compounds which can be used to modify the degradation of HMG-Coa reductase.
As such, in one aspect, the present invention provides isolated nucleic acids, i.e., polynucelotides, which encode the Hrd family of polypeptides. More particularly, the present invention provides an isolated nucleic acid which encodes a Hrd2p polypeptide, for example, the nucleic acid having the sequence set forth in SEQ ID NO:1. The present invention provides an isolated nucleic acid which encodes a Hrd3p polypeptide, for example, the nucleic acid having the sequence set forth in SEQ ID NO:2. In addition, the present invention provides an isolated nucleic acid encoding a polypeptide which is the human homologue of Hrd3p. Nucleic acids and corresponding polypeptides for Hrd1p are also provided. Vectors which include HRD nucleic acids are also a feature of the invention.
In another aspect, the present invention provides Hrd polypeptides which have the ability to modify the degradation of HMG-CoA reductase in a manner that is independent of the beneficial LDL receptor control axis. More particularly, the present invention provides a Hrd2 polypeptide which is encoded by the HRD2 nucleic acid having the sequence set forth in SEQ ID NO:1. The Hrd2p polypeptide is a soluble polypeptide having a molecular weight of about 109 kD with an amino acid sequence corresponding, for example, to SEQ ID NO: 3. In addition to Hrd2p, the present invention provides a Hrd3p polypeptide which is encoded, for example, by the HRD3 nucleic acid having the sequence set forth in SEQ ID NO:2. Hrd3p is a type I membrane polypeptide (i.e., the N-terminus is in the cytosol) having a molecular weight of about 99 kD, with a single membrane spanning sequence. An amino acid sequence of the polypeptide encoded by HRD3 is set forth in SEQ ID NO:4. It has been determined that when Hrd1p, Hrd2p and Hrd3p are supplied by trans complementation, they are able to restore Hrd1p-, Hrd2p- and Hrd3p-dependent HMG-CoA reductase degradation. In addition to the Hrd polypeptides isolated from yeast, the present invention provides the corresponding homologous human polypeptides. For example, the present invention provides a polypeptide which is the human homologue of Hrd3p. This polypeptide comprises amino acid sequences encoded by the nucleic acids set forth in SEQ ID NO:5 and SEQ ID NO:6. A Hrd3p human homologue subsequence is set forth in SEQ ID NO:8.
In one class of embodiments, the present invention provides a vector, such as a plasmid, virus or the like, which includes a nucleic acid that encodes a Hrd polypeptide, such as Hrd1p, a conservatively modified Hrd1p, Hrd2p, a conservatively modified Hrd2p, Hrd3p, or a conservatively modified Hrd3p polypeptide. Exemplar nucleic acid sequences for this purpose include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.
Isolated polypeptides encoded by the vectors, such as the polypeptides of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8; the Hrd1p sequence of FIG. 8 or the Hrd3p sequence of FIG. 8 are also provided.
In one group of embodiments, the invention provides vectors encoding one or more nucleic acids which hybridize under stringent conditions to a nucleic acid which encodes a polypeptide of the invention, such as the Hrd1p polypeptide, conservatively modified Hrd1p polypeptides, the Hrd2p polypeptide, conservatively modified Hrd2p polypeptides, the Hrd3p polypeptide, and a conservatively modified Hrd3p polypeptides. Exemplar nucleic acid subsequences include SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. Polypeptides encoded by such vectors are also a feature of the invention.
A class of vectors defined by the immunological reactivity of the encoded polypeptides are provided. The vectors include a nucleic acid that encodes a first polypeptide which is immunologically cross-reactive with a polypeptide of the invention, such as a full-length Hrd1p polypeptide, a full-length Hrd2p polypeptide, or a full-length Hrd3p polypeptide, wherein the first polypeptide, when supplied by trans complementation, restores HRD dependent HMG-CoA reductase degradation in a strain of yeast which does not produce a Hrdp polypeptide selected from the group consisting of Hrd1p, Hrd2p and Hrd3p (e.g., a strain which has a deletion in the gene encoding the specified protein). Exemplar polypeptides include SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8; the Hrd1p sequence of FIG. 8, (SEQ ID NO:25) and the Hrd3p sequence of FIG. 8 (SEQ ID NO:4).
In yet another aspect, the present provides a method for identifying a compound that modifies the degradation of HMG-CoA reductase, the method comprising: (i) providing a first solution comprising HMG-CoA reductase, a proteasome and a Hrd polypeptide; (ii) providing a second solution comprising HMG-Coa reductase, a proteasome, the Hrd polypeptide used in step (i) and a test compound; (iii) measuring the rate of HMG-CoA reductase degradation in the first solution and the second solution; and (iv) comparing the rate of HMG-CoA reductase degradation in the first solution with the second solution. If there is a difference between the rate of HMG-CoA reductase degradation in the first solution and the rate of HMG-CoA reductase degradation in the second solution, the compound tested is said to be capable of modifying the degradation of HMG-CoA reductase. In this method of the present invention, the HMG-CoA reductase can be provided in pure form or, alternatively, it can be provided in the form of a microsome or membrane fragment containing endogenous HMG-CoA reductase.
In a further aspect, the present invention provides another method of identifying a compound that modifies the degradation of HMG-CoA reductase, the method comprising: (i) applying the compound to be tested to a yeast strain containing elevated levels of HRD proteins; (ii) applying the compound to be tested to yeast strains containing normal levels of HRD proteins; and (iii) identifying the compounds that inhibit the growth of the first strains more than the growth of the second strains.
The present invention also provides methods of isolating a wild-type gene which regulates the degradation of HMG-CoA reductase, the methods comprising: (i) providing a yeast culture having a form of HMG-CoA reductase which is not regulated by the flux through the mevalonate pathway; (ii) selecting a yeast culture with increasing levels of resistance to a competitive inhibitor of HMG-CoA reductase; and (iii) cloning the wild type gene from a recombinant library by its ability to restore sensitivity to the competitive inhibitor of HMG-CoA reductase. Using these methods, the HRD nucleic acids of the present invention can be readily isolated from other eukaryotic sources. Moreover, using these methods, other members of the HRD family can be identified and isolated.
In another aspect, the present invention provides a method for isolating a wild type gene that encodes a protein involved in signalling for the degradation of HMG-CoA reductase, the method comprising: (i) providing a yeast culture having a form of HMG-CoA reductase whose degradation is low when flux through the mevalonate pathway is low; (ii) applying a mutagen to the culture; (iii) screening the culture for mutant cells that are more sensitive than the non-mutant parent strain to a competitive inhibitor of HMG-CoA reductase; and (iv) using a recombinant library to transform the mutant cells to identify clones capable of restoring to the cells resistance to the inhibitor of HMG-CoA reductase. Mutagens suitable for use in this method include, but are not limited to, chemical mutagens, physical mutagens (e.g., X-ray and the like) and transposons. Generally, the mutant cells of interest are more sensitive than the non-mutant parent strain to a competitive inhibitor of HMG-CoA reductase because they constitutively degrade the enzyme.
The present invention further provides kits for identifying a compound which modifies the degradation of HMG-CoA reductase, the kit comprising: a container, HMG-CoA reductase, a Hrd polypeptide and a proteasome. In addition, the present invention also provides a kit for isolating a gene which regulates the degradation of HMG-CoA reductase, the kit comprising a container and a yeast cell isolate which exhibits non-mevalonate dependent Hmg2p degradation. This particular kit may further contain an HMG-CoA reductase inhibitor. The kits of the present invention optionally include instructions for practicing the method.
Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description which follows.